NS-Seq

NS-Seq

Nascent strand sequencing (NS-Seq) sequences nascent DNA strands to locate DNA replication origins in the genome. NS-Seq utilizes lambda-exonuclease (λ-exo) to effectively digest parental DNA while leaving RNA-primer protected nascent strands intact. However, λ-exo inefficiently digests G-quadruplex structures (G4) and GC-rich motifs, but this bias can be normalized by using a λ-exo digested DNA from non-replicating cells as controls.

First, gDNA is enriched, made single-stranded, and 5'-phosphorylated using T4 polynucleotide K. Here, strands are digested with λ-exo and purified. Resultant single-stranded nascent strands are made double-stranded using random hexamers and fragmented to 100-600 bp sizes. DNA libraries are then prepared using standard kits and sequenced.

Pros:

• Locates DNA replication origins by sequencing RNA-primer protected nascent DNA strands
• λ-exo-digested DNA from non-replicating cells can be used as control to normalize for biases in lexo digestion
• Replacing K+ with Na+ during λ-exo digestion reduces digestion inefficiency with G4 regions

Cons:

• λ-exo does not efficiently digest G-quadruplex structures in plasmid and halts upon GC-rich motifs
• Purified samples can be contaminated with GC-rich and G4-protected DNA

• NS-Seq: Foulk M. S., Urban J. M., Casella C. and Gerbi S. A. (2015) Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res 25: 725-735

1. Smith O. K., Kim R., Fu H., Martin M. M., Lin C. M., et al. (2016) Distinct epigenetic features of differentiation-regulated replication origins. Epigenetics Chromatin 9: 18